Double FIT hybridization probes - towards enhancing brightness, turn-on and specificity of RNA detection.

Efficient fluorogenic hybridization probes combine high brightness and specificity of fluorescence signaling with large turn-on of fluorescence. Herein, we present an approach to enhance signaling by combining two identical fluorescence base surrogates in FIT2 probes. Provided there is a suitable positioning of dyes, target-bound FIT2 probes emit brighter than mono dye probes, while dye-dye contact in the single stranded state provides opportunities for decreasing background fluorescence. The probes were used to explore the single nucleotide-specific detection of a C → U edited RNA of the glycine receptor (GlyR). We observed strong self-quenching upon single base mismatched hybridization of FIT2 probes, which helped in distinguishing edited from unedited RNA target in cell lysates.

Prevalence of coat colour traits and congenital disorders of South American camelids in Austria, Germany and Switzerland.

BACKGROUND: The increasing popularity of alpacas and llamas outside of South America is undeniable. The associated limited genetic diversity raises questions about health and other genetically determined traits like coat colour. Therefore, a survey studying the prevalence of congenital disorders and coat colours and patterns in South American camelids was performed in Austria, Germany and Switzerland. Moreover, the motivation for keeping these animals, the herd size and breeds was assessed. RESULTS: A total of 146 questionnaires were returned corresponding to 16 farms from Austria, 69 farms from Germany, and 61 farms from Switzerland. In total, the returned surveys reported data on 2770 animals including ~ 85% alpacas and ~ 15% llamas. The most common alpaca breed was Huacaya (87.7%), the most common llama breed was Wooly (15.6%). Breeding (69.4%), wool production (63.3%) and keeping them as pets (53.7%) were the most common motivations to keep these animals, although this varied among countries. The three coat colour groups, solid white (24.8%), brown and black (64.8%) and grey (10.4%), occurred at different frequencies. About 7% of the South American camelids with solid white coat showed blue-pigmented eyes, corresponding to the known blue-eyed white phenotype, of which more than every second animal was apparently deaf. Uniform solid coloured animals occurred predominantly (81.4%), whereas pinto (8.8%), speckled (6.4%) and spotted (3.4%), also known as appaloosa, were comparably less prevalent. In total 161 observations of congenital disorders occurring during a 5-year-period were reported. The most prevalent disorders were in the group of musculoskeletal disorders such as spiral toe growth (16.4%), hyperextension of the fetlock joint (12.3%), angular limb deformities (11.0%) and axial rotation of the limbs (8.2%). CONCLUSIONS: This survey revealed first insights into the occurrence of different traits and disorders in the current South American camelid population of Austria, Germany, and Switzerland. The identification of the most common musculoskeletal disorders might encourage the breeders to eliminate affected animals from their breeding program to decrease the incidence although traits such as spiral toe growth might also represent phenocopies.

Chemo-biological mRNA imaging with single nucleotide specificity.

Unambiguous imaging of C → U edited mRNA calls for a method that distinguishes a locally high concentration of unbound probe or single nucleotide mismatched target from a locally low concentration of matched mRNA target. To address this issue, we combine FIT probes as a "chemical" detection system with the "biological" MS2 technique. Ratio measurements provide a convenient parameter to discriminate the edited from the unedited state of mRNA encoding for GlyR α2 in HEK cells.

Comparing Agent-Based Delivery of DNA and PNA Forced Intercalation (FIT) Probes for Multicolor mRNA Imaging.

Fluorogenic oligonucleotide probes allow mRNA imaging in living cells. A key challenge is the cellular delivery of probes. Most delivery agents, such as cell-penetrating peptides (CPPs) and pore-forming proteins, require interactions with the membrane. Charges play an important role. To explore the influence of charge on fluorogenic properties and delivery efficiency, we compared peptide nucleic acid (PNA)- with DNA-based forced intercalation (FIT) probes. Perhaps counterintuitively, fluorescence signaling by charged DNA FIT probes proved tolerant to CPP conjugation, whereas CPP-FIT PNA conjugates were affected. Live-cell imaging was performed with a genetically engineered HEK293 cell line to allow the inducible expression of a specific mRNA target. Blob-like features and high background were recurring nuisances of the tested CPP and lipid conjugates. By contrast, delivery by streptolysin-O provided high enhancements of the fluorescence of the FIT probe upon target induction. Notably, DNA-based FIT probes were brighter and more responsive than PNA-based FIT probes. Optimized conditions enabled live-cell multicolor imaging of three different mRNA target sequences.

Sequence-specific imaging of influenza A mRNA in living infected cells using fluorescent FIT-PNA.

Significant efforts have been devoted to the development of techniques allowing the investigation of viral mRNA progression during the replication cycle. We herein describe the use of sequence-specific FIT-PNA (Forced Intercalation Peptide Nucleic Acids) probes which contain a single intercalator serving as an artificial fluorescent nucleobase. FIT-PNA probes are not degraded by enzymes, neither by nucleases nor by proteases, and provide for both high sensitivity and high target specificity under physiological conditions inside the infected living host cell.

Remote control of lipophilic nucleic acids domain partitioning by DNA hybridization and enzymatic cleavage.

Lateral partitioning of lipid-modified molecules between liquid-disordered (ld) and liquid-ordered (lo) domains depends on the type of lipid modification, presence of a spacer, membrane composition, and temperature. Here, we show that the lo domain partitioning of the palmitoylated peptide nucleic acid (PNA) can be influenced by formation of a four-component complex with the ld domain partitioning tocopherol-modified DNA: the PNA-DNA complex partitioned into the ld domains. Enzymatic cleavage of the DNA linker led to the disruption of the complex and restored the initial distribution of the lipophilic nucleic acids into the respective domains. This modular system offers strategies for dynamic functionalization of biomimetic surfaces, for example, in nanostructuring and regulation of enzyme catalysis, and it provides a tool to study the molecular basis of controlled reorganization of lipid-modified proteins in membranes, for example, during signal transduction.

PNA FIT-probes for the dual color imaging of two viral mRNA targets in influenza H1N1 infected live cells.

Fluorogenic hybridization probes that allow RNA imaging provide information as to how the synthesis and transport of particular RNA molecules is orchestrated in living cells. In this study, we explored the peptide nucleic acid (PNA)-based FIT-probes in the simultaneous imaging of two different viral mRNA molecules expressed during the replication cycle of the H1N1 influenza A virus. PNA FIT-probes are non-nucleotidic, nonstructured probes and contain a single asymmetric cyanine dye which serves as a fluorescent base surrogate. The fluorochrome acts as a local intercalator probe and reports hybridization of target DNA/RNA by enhancement of fluorescence. Though multiplexed hybridization probes are expected to facilitate the analysis of RNA expression, there are no previous reports on the dual color imaging of two different viral mRNA targets. In this work, we developed a set of two differently colored PNA FIT-probes that allow the spectrally resolved imaging of mRNA coding for neuraminidase (NA) and matrix protein 1 (M1); proteins which execute distinct functions during the replication of the influenza A virus. The probes are characterized by a wide range of applicable hybridization temperatures. The same probe sequence enabled live-cell RNA imaging (at 37 °C) as well as real-time PCR measurements (at 60 °C annealing temperature). This facilitated a comprehensive analysis of RNA expression by quantitative (qPCR) and qualitative (imaging) means. Confocal laser scanning microscopy showed that the viral-RNA specific PNA FIT-probes neither stained noninfected cells nor cells infected by a control virus. The joint use of differently colored PNA FIT-probes in this feasibility study revealed significant differences in the expression pattern of influenza H1N1 mRNAs coding for NA or M1. These experiments provide evidence for the usefulness of PNA FIT-probes in investigations on the temporal and spatial progression of mRNA synthesis in living cells for two mRNA species.

Dual fluorophore PNA FIT-probes--extremely responsive and bright hybridization probes for the sensitive detection of DNA and RNA.

Fluorescently labeled oligonucleotides are commonly employed as probes to detect specific DNA or RNA sequences in homogeneous solution. Useful probes should experience strong increases in fluorescent emission upon hybridization with the target. We developed dual labeled peptide nucleic acid probes, which signal the presence of complementary DNA or RNA by up to 450-fold enhancements of fluorescence intensity. This enabled the very sensitive detection of a DNA target (40 pM LOD), which was detectable at less than 0.1% of the beacon concentration. In contrast to existing DNA-based molecular beacons, this PNA-based method does not require a stem sequence to enforce dye-dye communication. Rather, the method relies on the energy transfer between a "smart" thiazole orange (TO) nucleotide, which requires formation of the probe-target complex in order to become fluorescent, and terminally appended acceptor dyes. To improve upon fluorescence responsiveness the energy pathways were dissected. Hydrophobic, spectrally mismatched dye combinations allowed significant (99.97%) decreases of background emission in the absence of a target. By contrast, spectral overlap between TO donor emission and acceptor excitation enabled extremely bright FRET signals. This and the large apparent Stokes shift (82 nm) suggests potential applications in the detection of specific RNA targets in biogenic matrices without the need of sample pre-processing prior to detection.

FIT probes: peptide nucleic acid probes with a fluorescent base surrogate enable real-time DNA quantification and single nucleotide polymorphism discovery.

The ability to accurately quantify specific nucleic acid molecules in complex biomolecule solutions in real time is important in diagnostic and basic research. Here we describe a DNA-PNA (peptide nucleic acid) hybridization assay that allows sensitive quantification of specific nucleic acids in solution and concomitant detection of select single base mutations in resulting DNA-PNA duplexes. The technique employs so-called FIT (forced intercalation) probes in which one base is replaced by a thiazole orange (TO) dye molecule. If a DNA molecule that is complementary to the FIT-PNA molecule (except at the site of the dye) hybridizes to the probe, the TO dye exhibits intense fluorescence because stacking in the duplexes enforces a coplanar arrangement even in the excited state. However, a base mismatch at either position immediately adjacent to the TO dye dramatically decreases fluorescence, presumably because the TO dye has room to undergo torsional motions that lead to rapid depletion of the excited state. Of note, we found that the use of d-ornithine rather than aminoethylglycine as the PNA backbone increases the intensity of fluorescence emitted by matched probe-target duplexes while specificity of fluorescence signaling under nonstringent conditions is also increased. The usefulness of the ornithine-containing FIT probes was demonstrated in the real-time PCR analysis providing a linear measurement range over at least seven orders of magnitude. The analysis of two important single nucleotide polymorphisms (SNPs) in the CFTR gene confirmed the ability of FIT probes to facilitate unambiguous SNP calls for genomic DNA by quantitative PCR.

Synthesis and functional studies of THF-gramicidin hybrid ion channels.

THF-gramicidin hybrids 2-4 with the L-THF amino acid 1 in positions 11 and 12 and compounds 5-8 with the D-THF amino acid ent-1 in positions 10 and 11 were synthesized and their ion channel properties were studied by single-channel-current analysis. The replacement of positions 11 and 12 by the L-THF amino acid 1 gave a strongly reduced channel performance. In contrast, replacement of positions 10 and 11 by the D-THF amino acid ent-1 gave rise to new and interesting channel properties. For the permeability ratios, the ion selectivity shifts from Eisenman I towards Eisenman III selectivity and the channels display ms-dynamics. Most remarkable is the asymmetric compound 8, which inserts selectively into a DPhPC membrane and displays voltage-directed gating dynamics.

Electrophysiological response of cultured trabecular meshwork cells to synthetic ion channels.

The response of living cells of the trabecular meshwork to synthetic ion channels is described. The THF-gramicidin hybrids THF-gram and THF-gram-TBDPS as well as a linked gA-TBDPS and gramicidin A were applied to cultured ocular trabecular meshwork cells. THF-gram application (minimal concentration, 10(-8) M; saturation, 10(-7) M) led to an additional conductance which displayed characteristics of weak Eisenman-I-selective cation channels, no cell destruction, an asymmetric change of the inward/outward currents, and higher current densities using Cs(+) as charge carrier compared to Na(+) and K(+). Linked-gA-TBDPS showed at 10(-12) M increases of the membrane conductance comparable to gA at 10(-7) M and a much faster response of the cells. Thus, THF-gramicidin hybrids form a basis for the use of synthetic ion channels in biological systems, which eventually may lead to new therapeutic approaches.

Cyclohexylether δ-Amino Acids: New Leads for Selectivity Filters in Ion Channels.

Synthetic ion channels containing δ-amino acids can become cation selective! δ-Amino acids with a cyclohexylether unit were combined with structural motives from gramicidin A and led to channels with a NH4+ /K+ permeability ratio of >10/1. (The current trace for an NH4+ channel is shown.).

Tentativa de suicídio e adesão ao tratamento: um estudo descritivo em hospital geral / Attempted suicide and treatment compliance: a descriptive study in the general hospital

Dados sócio-demográficos e clínicos foram coletados de forma padronizada em uma amostra consecutiva de 156 casos de tentativa de suicídio (TS) atendidos no pronto-socorro de um hospital geral universitário. Um programa de atendimento para esses pacientes incluiu a constituição de uma equipe multidisciplinar e estratégias para o tratamento ambulatorial. A adesão ao tratamento foi verificada após três meses da TS. Foram atendidos 110 (70,5 por cento) mulheres e 46 (29,5 por cento) homens, apresentando idade média de 27 anos e com 2/3 dos casos abaixo de 30 anos. Geralmente a TS foi ato impulsivo, com baixa intencionalidade suicida. Excesso de medicamentos e ingestão de venenos foram utilizados em 60 por cento e 20 por cento dos casos, respectivamente. Desentendimento com pessoa próxima ocorrido no último mês foi relatado por 70 por cento dos pacientes, funcionando como desencadeante da TS. Alguns pacientes já haviam passado por tratamento (35 por cento) e internação (15 por cento) psiquiátricos. O índice médio de comparecimento à primeira consulta ambulatorial foi de 55 por cento, sendo maior quando havia facilidade de transporte (65-78 por cento) e história de tratamento (68 por cento) e internação (79 por cento) psiquiátricos. Após três meses da TS, o abandono do tratamento chegou a 59 por cento. Estratégias para se aumentar a adesão através do correio não foram eficazes. Ao final desse período, os casos que permaneciam em tratamento em nosso serviço eram em sua maioria de transtornos psiquiátricos estabelecidos, semelhantes aos de um ambulatório psiquiátrico geral

Tentativa de suicidio e adesao ao tratamento: um estudo descritivo em hospital geral

Dados socio-demograficos e clinicos foram coletados de forma padronizada em uma amostra consecutiva de 156 casos de tentativa de suicidio (TS) atendidos no pronto-socorro de um hospital geral universitario. Um programa de atendimento para esses pacientes inclui a constituicao de uma equipe multidisciplinar e estrategias para o tratamento ambulatorial. A adesao ao tratamento foi verificada apos tres meses da TS. Foram atendidos 110(70,5 por cento) mulheres e 46(29,5 por cento) homens, apresentando idade media de 27 anos e com 2/3 dos casos abaixo de 30 anos. Geralmente a TS foi ato impulsivo, com baixa intencionalidade suicida. Excesso de medicamentos e ingestao de venenos foram utilizados em 60 por cento e 20 por cento dos casos, respectivamente. Desentendimento com pessoa proxima ocorrido no ultimo mes foi relatado por 70 por cento dos pacientes, funcionando como desencadeante da TS. Alguns pacientes ja haviam passado por tratamento (35 por cento) e internacao (15 por cento) psiquiatricos. O indice medio de comparecimento a primeira consulta ambulatorial foi de 55 por cento, sendo maior quando havia facilidade de transporte (65-78 por cento) e historia de tratamento (68 por cento) e de internacao (79 por cento) psiquiatricos. Apos tres meses da TS, o abandono do tratamento chegou a 59 por cento. Estrategias para se aumentar a adesao atraves do correio nao foram eficazes. Ao final desse periodo, os casos que permaneciam em tratamento em nosso servico eram em sua maioria de transtornos psiquiatricos estabelecidos, semelhantes aos de um ambulatorio psiquiatrico geral.